Mycobacteria Flashcards
important human pathogens of Mycobacteria
○ Mycobacterium tuberculosis complex (MTBC)
○ Nontuberculosis mycobacteria (NTM)
○ Mycobacterium avium-intracellulare complex (MAI)
What are mycobacteria considered?
○ BSL 3
○ recommended .to reduce the risk of inhalation through aerosolization
Describe Mycobacterium
○ Slender, slow growing bacilli
○ Obligate aerobes
○ Cell division occurs by branching
How do you enhance growth of Mycobacterium?
Growth enhanced with CO2 (10%)
What is in the cell wall of Mycobacterium?
○ ~60% of the cell wall is comprised of lipids and lipid containing structures
○ Mycolic acid and Cord factor
Mycolic acid
○ strong hydrophobic molecules that form a lipid shell around the organism
○ affect permeability properties at the cell surface
What is Mycolic acid an important virulence factor for?
Mycobacterium tuberculosis
Cord Factor
○ responsible for the serpentine cording
○ toxic to mammalian cells
○ an inhibitor of PMN migration
Where is cord factor main made?
virulent strains of Mycobacterium tuberculosis
What do the high concentration of lips contribute to Mycobacterium tuberculosis?
○ Resistance to many antibiotics
○ Resistance to killing by acidic and alkaline compounds
○ Resistance to osmotic lysis
○ Resistance to lethal oxidations and survival inside of macrophages
○ Impermeability to stains and dyes
Acid-fast bacilli (AFB)
Mycobacteria that are difficult to decolorize when using as acid alcohol
What are the 3 staining methods for mycobacteria?
○ Ziehl-Neelsen technique
○ Kinyoun Technique
○ Fluorochrome Stain
Ziehl-Neelsen technique
○ smear is fixed and stained with hot carbol-fuchsin
○ decolorized with acid-alcohol
○ counterstained with methylene-blue
○ Acid fast bacilli appear pink in a contrasting background
○ Also this is no longer used
Kinyoun Technique
○ Similar to Ziehl-Neelsen technique but uses detergents to drive the dye into the cell wall instead of heat
○ Similar in appearance (pink contrast to background)
Fluorochrome Stain
○ uses auramine O as a primary stain
○ rhodamine as the counter stain
Fluorochrome stain fact
○ stains are more sensitive
○ more labor intensive and must be read using a fluorescent microscope
mycobacteria collection?
○ Sputums
○ Bronchial washes
○ Transtracheal aspirates
When do you collect mycobacteria samples? How many?
○ 3 samples
○ early morning preferred
○ for 3 consecutive days
What if a sample collection of sputum isn’t available? Like in children
Gastric lavage, early in the morning
What is done to urine samples of mycobacteria?
Centrifuged to concentrate
What fluids are collected for mycobacteria?
○ CSF
○ synovial fluid
○ pericardial fluid
○ peritoneal fluid
For blood samples, what is used to release the bacteria from red/white blood cells?
Lysing agents (saponin)
What is needed for mycobacteria processing of respiratory samples?
Decontamination and digestion
What is used for decontamination? What does it do?
○ NaOH or benzalkonium chloride
○ Kills normal respiratory flora or pathogens
○ reduces contaminants
How does NaOH work?
increases pH to a level that is antibacterial
What is used for digestion? What does it do?
○ N-acetyl-L-cysteine (NALC)
○ Liquefies mucus in a respiratory sample which may trap mycobacteria
What is needed to grow mycobacteria?
whole egg
What is the traditional growth media for mycobacteria?
Lowenstein-Jensen Media
Describe Lowenstein-Jensen Media
○ Prepared as agar slants
○ Malachite green (inhibitor)
○ Incubation 6-10 weeks
○ Can see growth as early as 18-24 days
What is the agar based media to grow mycobacteria?
Middlebrook 7H10 or Middlebrook 7H11
Describe Middlebrook 7H10/7H11
○ transparent
○ contains 2% glycerol to support the growth of MAC
○ Growth detected between 10-12 days
What is Middlebrook 7H9 broth?
○ nonselective liquid media
○ maintains stock cultures
○ prepares isolates for biochemical analysis
What is Mycobacterium growth index tube (MGIT)
○ modified 7H9 broth with a fluorchrome
○ Glows under UV light if positive
What are the incubation temperatures for mycobacteria species?
○ MTB 35°C – 37°C
○ M. marinum and M. ulcerans 30°C - 32°C
○ M. xenopi 42°C – 45°C
Are what CO2 level are mycobacterium kept at?
10%
What does Mycobacterium Tuberculosis cause?
As the name implies, tuberculosis
How is Mycobacterium Tuberculosis transmitted?
respiratory droplets containing the organism
Once it’s inhaled, where does Mycobacterium Tuberculosis deposit?
bronchiole or alveoli
For immunocompromised patients, where does Mycobacterium Tuberculosis spread?
○ lymph nodes
○ Spine
○ long bone
○ Heart
○ Meninges
○ gastro-urinary system
Mantoux tuberculin skin test (TST)
○ used to determine if a person has been exposed to MTB organisms
○ Uses PPD that is injected into the forearm
or Mantoux tuberculin skin test (TST), what causes false positive reactions?
Calmette-Guerin (BGC) vaccination
For Mantoux tuberculin skin test (TST), what causes false negative reactions?
○ Suppressed immune system can cause skin test to not react
○ Recent or very old infection
○ Infants less than 6 months of age with an overwhelming TB disease
For TST what is recommended for healthcare workers?
○ Two step testing is recommended
○ second TST is administered if first is negative after 1-3 weeks
○ If both tests are negative, person is fine
QuantiFERON TB Gold In-Tube test (QFT-GIT)
Looks for antibodies produced from past or present infection of TB
T-SPOT TB test (T-Spot)
○ Blood tests to detect TB infection
○ Can detect latent TB (nonactive TB)
How does T-SPOT TB test (T-Spot) work?
When WBCs are infected with MTB a substance interferon – gamma (IFN-γ) is released and can be detected
For MTB, what are some identifiers on LJ or Middlebrook media?
○ tan to buff colonies in 14-28 days at 35-37°C
○ Colony morphology is rough, dry, warty and granular (looks like breadcrumbs or cauliflower)
What are some 1st line drugs against MTB?
○ Isoniazid (INH)
○ Rifampin
○ Pyrazinamide
○ Ethambuto
What are some 2nd line drugs against MTB?
○ Streptomycin
○ Cycloserine
○ P-aminosalicylic acid
○ Ethionamide
What is the mycobacterium tuberculosis complex?
Genetically related group of Mycobacterium species that can cause tuberculosis in humans or other organisms
What organisms are a part of the mycobacterium tuberculosis complex?
○ M. tuberculosis
○ M. africanum
○ M. canettii
○ M. bovis
○ M. microti
○ M. caprae
○ M. orygis
○ M. pinnipedii
Mycobacterium avium complex (MAC) consists of two species:
M. avium and M. intracellulare
Why are the two species of MAC also called Mycobacterium avium-intracellulare (MAI)?
Because these species are difficult to differentiate
Mycobacterium avium-intracellulare (MAI)
atypical Mycobacterium most commonly associated with human disease
MAC is primarily a what?
○ pulmonary pathogen
○ affects individuals who are immune compromised
How would you Identify MAC group?
○ acid-fast bacillus (AFB)
○ Colony morphology is smooth
○ tan to buff colonies in 14-28 days at 35-37°C
M. ulcerans
○ produces cutaneous lesions called Buruli ulcers
○ Appear as lumps under the skin
○ non-healing ulcers
M. ulcerans identification?
Flat, rough, pale colonies on LJ and Middlebrook agars at 32-33°C
M. bovis
Agent of tuberculosis in cattle and other warm-blooded animals
How has M. bovis been handled?
○ better pasteurization techniques
○ strict guidelines on cattle handling and management by FDA
Bacillus Calmette–Guérin (BCG) vaccine
vaccine primarily used against tuberculosis in countries where tuberculosis is common
M. leprae
Cause of Hansen’s disease or leprosy
How do you grow M. leprae? And why?
○ grown on the foot pads of mice
○ Cannot be cultured in vitro
M. Leprae Characterized by?
○ skin sores and nerve damage
○ Can get worse without treatment:
lesions on the skin
numbness in the extremities
muscle weakness
M. leprae Identification?
AFB in characteristic skin lesion specimens
